title: A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.
abstract: Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.
authors.forename: Amita
authors.initials: A
authors.lastname: Gupta
authors.forename: Nimisha
authors.initials: N
authors.lastname: Shrivastava
authors.forename: Payal
authors.initials: P
authors.lastname: Grover
authors.forename: Ajay
authors.initials: A
authors.lastname: Singh
authors.forename: Kapil
authors.initials: K
authors.lastname: Mathur
authors.forename: Vaishali
authors.initials: V
authors.lastname: Verma
authors.forename: Charanpreet
authors.initials: C
authors.lastname: Kaur
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Chaudhary
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000276:immunology; D016366:Open Reading Frames / Q000235:genetics; D019151:Peptide Library; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D019151:Peptide Library; D014357:Trypsin